The ability of long non-coding RNA RP11-284N8.3 to predict the risk, the severity and 28-day mortality of adults with sepsis

In a prior study, we identified a novel sepsis specific long noncoding RNAs (lncRNA) RP11-284N8.3, which may primarily participate in T cell activation and immune response during sepsis. However, the clinical significance of lncRNA RP11-284N8.3 in sepsis remains entirely unknown. This single-center prospective cohort study enrolled 147 adults with sepsis and 74 healthy controls (HCs) with matched age and sex between January 2021 and November 2022 at our hospital. Blood samples and clinical data were collected from HCs at enrollment and from adults with sepsis within 24 hours after admission. lncRNA RP11-284N8.3 expression was detected by RT-qPCR. The relative expression of lncRNA RP11-284N8.3 was significantly decreased in adults with sepsis compared to HCs (P < .0001), in adults with septic shock compared to adults without shock (P = .0012), and in 28-day deaths compared to 28-day survivors (P = .0006). receiver operating characteristic curves of lncRNA RP11-284N8.3 in predicting sepsis severity and 28-day mortality showed an area under the curve of 0.6570 (95% confidence interval [CI]: 0.5701–0.7440) and an area under the curve of 0.6765 (95% CI: 0.5809–0.7721), respectively. Multivariate logistic regression analysis revealed that lncRNA RP11-284N8.3 was an independent risk factor for 28-day mortality in adults with sepsis (odds ratio: 0.1057, 95% CI: 0.0115–0.7746, P = .0328). Low expression of lncRNA RP11-284N8.3 is correlated with increased risk, severity and 28-day mortality in adults with sepsis, and it may function as a potential biomarker to facilitate the diagnosis and management of sepsis.


Introduction
Sepsis is defined as a life-threatening multiorgan dysfunction caused by a dysregulated host response to infection. [1] Annually, approximately 48.9 million population are affected by sepsis, over 11 million of those affected die, and one 6th of sepsis survivors experience significant functional limitations. [2][3][4] In view of the high morbidity, disability and mortality, sepsis has now been established as the quintessential medical disorder of the twenty-first century. [5] Recent decades have seen the remarkable progress of treatments for sepsis, including fluid therapy, vasoactive drug, antimicrobial agent, etc. Unfortunately sepsis remains the most common cause of death in the intensive care unit (ICU) due to delayed diagnosis and inadequate management in the initial hours. Under such a background, an urgent investigation into novel biomarkers contributing to identify the risk of sepsis timely and monitor the prognosis of sepsis precisely would be significant.
Over the past decade, long noncoding RNAs (lncRNAs) have become an active area of sepsis research. lncRNAs are a group of nonprotein-coding transcripts with more than 200 nucleotides in length [6] and have been reported to exert key regulatory roles in the pathogenesis of various diseases. [7][8][9][10][11] With the advance of high throughput sequencing technology, an increasing number of sepsis-associated lncRNAs had been identified. [12][13][14][15][16] In our prior study, [17] the lncRNA-mRNA network in sepsis was constructed based on a publicly available RNA-seq data, and we identified a potential key sepsis-associated lncRNA RP11-284N8. 3. At present, lncRNA RP11-284N8.3 is largely with unknown functions in sepsis. However, we found most mRNAs highly co-expressed with lncRNA RP11-284N8.3 primarily enriched in immune response and T Medicine cell activation related Gene Ontology biological process terms. Our results indicated that the novel sepsis specific lncRNA RP11-284N8.3 may exert potential roles in regulating immune response during sepsis and may be closely related to the development and progression of sepsis. Nonetheless, the clinical significance of lncRNA RP11-284N8.3 in sepsis patients remains largely unknown.
In the present study, we aim to investigate the expression of lncRNA RP11-284N8.3 from peripheral blood monouclear cells (PBMCs) with risk, severity and 28-day mortality in adults with sepsis. The findings may contribute to the understanding of lncRNA RP11-284N8.3 in sepsis and provid a new insight into sepsis management.

Study population
This was a single-center prospective cohort study performed in Henan Provincial People's Hospital. A total of 173 sepsis patients were admitted to our emergency intensive care unit within the initial 24 hours from January 2021 to November 2022. According to the "sepsis-3" consensus, [1] all patients were diagnosed with sepsis. Patients were excluded if they were aged under 18 years or over 80 years, transferred from another hospital, receiving immunosuppressive therapy, concomitant with preexisting autoimmune diseases, suffering from malignancies, or in pregnancy or lactation. Within that same time frame, 73 healthy individuals with matched sex and age features were chosen as healthy controls (HCs), who all underwent physical examination in our hospital.
The present study was approved by the ethics committee of Henan Provincial People's Hospital (2021-49). The written informed consent was obtained from all participant or their guardians.

Clinical data collection
The clinical data of all enrolled adults with sepsis were obtained from clinical electronic medical records, including demographics (age, gender, and smoke), chronic comorbidities, laboratory data measured at admission (white blood cell, blood platelet, C-reactive protein [CRP], procalcitonin [PCT], total bilirubin, serum creatinine, albumin, serum electrolyte, arterial lactic acid), and primary infection site, disease severity (SOFA score, APACHE II score, septic shock) assessed by a senior doctor. Patients were followed up until death or 28 days after ICU admission, and 28-day mortality was calculated as well. Based on the survival status in 28-day follow up, sepsis patients were further classified as 28-day survivors and 28-day deaths. In addition, the demographics and laboratory data of HCs were also recorded at enrollment.

Sample collection and qRT-PCR detection
Peripheral blood samples were collected from HCs at enrollment and from sepsis patients within 24 hours after admission. All the blood samples were processed immediately after collection for the isolation of PBMCs, which were stored at − 80°C before RNA extraction. Total RNA was extracted from PBMCs using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then, the purified RNA was reverse-transcribed taken for complementary DNA by PrimeScript RT reagent Kit (Takara). Subsequently, qRT-PCR was conducted by using TB Green Fast qPCR Mix (Takara) and specific primers (forward: 5ʹ-GTCCTCCACTAATCACAGAAT-3ʹ, reverse: 5ʹ-TCACTT-GATGTCAGAATGCT-3ʹ). Relative gene expression was determined by employing the 2 −ΔΔCT method and normalized against GAPDH.

Statistical analysis
All statistical analyses were carried out using GraphPad Prism 9.0 software (GraphPad Software lnc., San Diego, California). Continuous variables were presented as mean ± standard deviation or median and inter-quarter range, as appropriate, depending on the normality of the data, which was assessed using Kolmogorov-Smirnov test. Normally distributed continuous variables were compared using Student t test, while the continuous variables that were not normally distributed were compared using Mann-Whitney U test. Categorical variables were present as counts and proportions, and were tested using Chi-square test or Fisher exact test. Correlation analysis was determined by Spearman rank correlation test. The receiver operating characteristic (ROC) curve analysis was used to determine the predictive accuracy of lncRNA RP11-284N8.3 in predicting the risk, severity and 28-day mortality of sepsis, and the area under the curve (AUC) (odds ratio [OR], 95% confidence interval [CI]) was calculated. Logistic regression analysis was used to determine 28-day mortality-related factors. After univariate analysis, variables with P value < .10 were included in multivariate regression analysis. A 2-sided P value < .05 was considered statistically significant.

Baseline and clinical characteristics of HCs and adults with sepsis
A total of 173 adults with sepsis admitted to our emergency intensive care unit within the initial 24 hours between January 2021 and November 2022 were consecutively enrolled. Of whom, 26 adults were excluded as their family members quitted aggressive treatment or rescue for economic reasons. Finally, 147 adults with sepsis and 73 HCs were analyzed in this study.
Baseline and clinical characteristics of HCs and adults with sepsis can be viewed in Table 1. The median age of sepsis patients was 62 (49, 71) years, and there were 87 (59.18%) males. Thirty (20.41%) patients had a history of smoking. No difference of age (P = .2277), gender (P = .3148) and history of smoking (P = .4805) between sepsis patients and HCs was observed. Fortythree (29.25%) had a chronic comorbidity of hypertension, and 55 (37.41%) had a primary respiratory infection. Regarding laboratory results at admission, increased levels of white blood cell (P < .0001), CRP (P < .0001), PCT (P < .0001) and serum creatinine (P < .0001), while decreased levels of blood platelet (P = .0287) and albumin (P = .0003) were observed in sepsis patients compared to HCs. The mean lactic acid level in sepsis patients was 1.64 ± 1.09 mmol/L. The levels of total bilirubin (P = .6147), sodium (P = .8363), potassium (P = .8869), and calcium (P = .6993) did not differ significantly between sepsis patients and HCs. Regarding sepsis severity score, APACHE II score and SOFA score showed a mean value of 18.77 ± 3.08 and 10.71 ± 2.28, respectively.

Discussion
lncRNAs have been demonstrated to exert key regulatory roles in all sorts of biological processes, thereby contributing to the development and progression of diverse diseases, including sepsis. [18,19] In our prior study, [17] we profiled the differentially expressed lncRNAs and mRNAs co-expression network in sepsis based on a publicly available RNA-seq data, and identified 3 sepsis specific lncRNAs, including RP11-284N8.3, CTB-61M7.2, and LINC00861. Among of them, lncRNA RP11-284N8.3 is entirely unknown to sepsis and may primarily participate in T cell activation and immune response based on the functional enrichment analysis of its highly co-expressed mRNAs. As we all know, sepsis initiates a complicated immunopathogenesis process that involves both innate and adaptive immune responses. [20,21] Thus, there is no doubt that lncRNA RP11-284N8.3 is associated with the occurrence and development of sepsis.
Prior studies have revealed that lncRNAs may function as potential biomarkers for the diagnosis and prognosis of sepsis, such as the well-known lncRNAs NEAT1 and MALAT1. [14,22,23] Herein, we discovered that the relative expression of lncRNA RP11-284N8.3 was remarkably decreased in adults with sepsis compared to that in HCs, which suggested that lncRNA RP11-284N8.3 were closely correlated with sepsis. Additionally, we explored the correlation of lncRNA RP11-284N8.3 with disease severity in adults with sepsis. It is well-known that APACHE II score and SOFA score have become the most accepted and used among the general ICU severity of illness scoring systems. [24] We found that lncRNA RP11-284N8.3 was negatively correlated with APACHE II score and SOFA score. Septic shock is the most severe form of sepsis and is a state of circulatory failure that occurs in a subset of sepsis patients. [25] Lower expression of lncRNA RP11-284N8.3 were also observed in adults with septic shock compared to adults without septic shock. Taken together, lncRNA RP11-284N8.3 can serve as a novel biomarker for predicting sepsis risk and severity.
Besides, we also found that the relative expression lncRNA RP11-284N8.3 was decreased in 28-day deaths compared to 28-day survivors, and it was an independent factor with a moderate degree of predictive value for 28-day mortality risk in adults with sepsis (AUC: 0.6765, 95% CI: 0.5809-0.7721). The value was numerically similar to that of CRP and lactic acid, but was weaker than that of APACHE II score and SOFA score. Notably, ROC curve showed that the combination of lncRNA RP11-284N8.3, CRP, lactic acid, APACHE II score and SOFA score could well-predict 28-day mortality risk in adults with sepsis, suggesting that it might be a potential tool to recognize sepsis patients who have a high 28-day mortality risk, which may improve the management toward these patients.
Despite the interesting results, the limitations of this study are as follows: First, the relatively small sample size might result in Table 2 Baseline, clinical characteristics and lncRNA RP11-284N8.3 relative expression in survivors and deaths groups. .0006 CRP = C-reactive protein, IQR = inter-quarter range, PCT = procalcitonin, PLT = blood platelet, SD = standard deviation, Scr = serum creatinine, TBil = total bilirubin, WBC = white blood cell.

Conclusions
To the best of our knowledge, this is the first study to evaluate the predictive value of lncRNA RP11-284N8.3 for risk, severity and 28-day mortality in adults with sepsis. Our results demonstrated that lncRNA RP11-284N8.3 was down-regulated in sepsis patients and was negatively correlated with sepsis severity and possessed a moderate predictive value on 28-day mortality risk. LncRNA RP11-284N8.3 could serve as a potential biomarker for facilitating diagnosis and management in sepsis patients.